Methods for assessing fatigue level and applications thereof

ABSTRACT

Level of fatigue that accompanies everyday life or a disease can be simply, easily, and quantitatively assessed by obtaining a body fluid from a test subject and measuring the amount of human herpesvirus in the body fluid. Furthermore, the anti-fatigue potency of anti-fatigue substances and anti-fatigue food products can be measured.

TECHNICAL FIELD

The present invention relates to methods for assessing human fatiguelevel and methods of application thereof, kits for assessing fatiguelevel, and methods for measuring anti-fatigue activity of anti-fatiguesubstances.

BACKGROUND ART

Fatigue is a very familiar problem in everyday life. Many modern peoplewho are under a lot of stress suffer from various kinds of fatigue.However, scientific and/or medical studies relating to “fatigue” haveonly been fragmentarily conducted, and hardly any studies have been doneon decisive means or quantitative standards for quantitatively andobjectively expressing “fatigue”, which is a subjective symptom.

So far, muscle fatigue (exercise fatigue) has been mainly studied as arepresentative example of “fatigue”. The indicator focused on in thiscase is an increase in the production of lactic acid in muscles.However, lactic acid is basically an important energy source for thecranial nervous system, and the theory that it inhibits muscle activityis now negatively perceived. In addition, during muscle fatigue, thephenomena of increase of pyruvic acid and drop of pH value in bodyfluids are known to occur. These phenomena are indeed observed when acertain stress, a load to muscle (exercise load), is given; however“fatigue” is different from local muscle exhaustion and is considered tobe a broader and larger physiological phenomenon that manifests in theliving body.

Patent Document 1 discloses a method for quantifying stress by using asindicators concentrations of adrenal sex steroids and their metabolitesin saliva. Patent Documents 2 and 3 disclose a method for assessing theanti mental fatigue activity of test substances by measuring amino acidssuch as taurine, leucine, and isoleucine in blood. Although thesedocuments disclose methods for assessing stress or fatigue by usingbiological factors in body fluids as indicators, the quantification offatigue in everyday life is yet to be achieved.

Immune strength is thought to deteriorate when humans are fatigued, andvirus infection can be given as one form of expression of thisdeterioration of human immune strength. However, a relationship betweenfatigue and virus infection in humans has not been elucidated.

Eight species have so far been identified as herpesviruses that mainlyinfect humans. Herpesviruses are large DNA viruses. They are classifiedinto three subfamilies α, β, and γ, mainly according to the phylogenetictree of evolution, and each subfamily has common biologicalcharacteristics. For instance, α-herpesvirus is neurotropic andundergoes latent infection and reactivation in nerve cells, andγ-herpesvirus has tumorigenicity.

Characteristics common to all herpesviruses include establishment oflatent infection by viruses hiding within the body following mainlychildhood infections, and recommencement of growth following somestimuli (reactivation).

Fatigue, especially mental fatigue and stress, is cited as a possiblestimulus that induces reactivation. Among herpesviruses, herpes simplexvirus type 1 is the causative of labial herpes. The reactivation of thisvirus is often observed as manifestation of labial herpes, which happensin many cases when virus carriers become extremely exhausted.

However, it actually remains almost unknown as to the sort of stimulusthat induces the reactivation of herpesviruses. The relationship betweenthe reactivation of herpesviruses and fatigue/stress has not beenscientifically proven, and the above-mentioned relationship betweenherpes simplex virus type 1 and fatigue is merely a folk belief.

As mentioned above, methods for objectively judging muscle fatigue(exercise fatigue) due to muscle load (exercise load) have beenproposed. However, hardly any reports exist regarding an objectivemethod for assessing fatigue symptoms of everyday life although manymodern people are experiencing them. Fatigue symptoms of everyday life,if they are ignored, carry the risk of directly resulting in death fromoverwork, a sudden death caused by long-term overworking. Although theproblem of overwork death is recognized as very important from medical,economic, and social standpoints, scientific mechanisms underlying itare hardly clarified. Therefore, an objective method for assessingfatigue level is required in order to prevent overwork death, which hasrecently become a social problem.

Furthermore, since most medicines, health foods, or such, includingnutrition-supplement drinks that are flooding the market, advertisefatigue recovery or prevention functions, scientific evidence regardingthe functionality is widely required not only by consumers, but also bythe market and society as a whole.

As mentioned above, knowledge on fatigue caused by exercise load ispresent. However, fatigue caused by exercise load and fatigue ineveryday life are completely different from each other, and a method forassessing fatigue in everyday life has not been developed. Thus, thedevelopment of a simple, easy, and objective in vivo method forassessing fatigue in everyday life and their methods of application arestrongly required.

[Patent Document 1]

Japanese Patent Application Kokai Publication No. (JP-A) Hei 11-038004(unexamined, published Japanese patent application)

[Patent Document 2] JP-A Hei 11-304792 [Patent Document 3] JP-A Hei11-304793 DISCLOSURE OF THE INVENTION Problems to be Solved by theInvention

An objective of the present invention is to provide methods for simply,easily, and quantitatively assessing fatigue level, methods ofapplication thereof, kits for assessing fatigue level, and methods formeasuring anti-fatigue potency of anti-fatigue substances.

Means for Solving the Problems

As a result of dedicated investigations in view of the above problems,the present inventors made the original discovery that levels of fatiguecaused in everyday life can be quantitatively assessed by measuring andassessing the amount of human herpesvirus (HHV) in a body fluid, andcompleted the present invention that enables the measurement of fatiguelevels in everyday life by utilizing this experimental system.

Namely, the present invention's methods for assessing fatigue level arecharacterized by assessing fatigue levels using, as an indicator, theamount of at least one species of HHV in a body fluid, in order to solvethe above problems. The above-mentioned methods can simply, easily, andobjectively assess the degree of fatigue in humans, and can alsoquantitatively estimate an effect and efficacy of medicines having theeffect of restoring or preventing fatigue, as well as that ofnutritional supplement food products such as nutrition-supplement drinksor health foods. Furthermore, an overworked state, which is apt to becaused by long hours of work and such, can be simply, easily, andobjectively detected.

The kits for assessing fatigue level according to the present inventionare characterized by being used to carry out the above-mentioned methodsfor assessing fatigue level in order to solve the above-mentionedproblems.

The methods for assessing anti-fatigue potency of anti-fatiguesubstances according to the present invention are characterized bymeasuring anti-fatigue potency of anti-fatigue substances with eitherthe above-mentioned methods for assessing fatigue level or the kits forassessing fatigue level, in order to solve the above-mentioned problems.

Namely, in order to solve the above-mentioned problems, the followinginventions are specifically provided:

[1] A method of assessing fatigue level, comprising assessing fatiguelevel of a test subject using human herpesvirus amount in a body fluidas an indicator,[2] The method of assessing fatigue level of [1], wherein the fatiguelevel is assessed to be high when the amount of human herpesvirus ishigh.[3] The method of assessing fatigue level of [2], wherein the amount ofhuman herpesvirus is measured as an expression level of a gene of humanherpesvirus.[4] The method of assessing fatigue level of any one of [1] to [3],wherein the method comprises assessing the test subject to be in a stateof overwork due to an accumulation of medium- or long-term acute fatiguearising from everyday life, when the amount of human herpesvirus ishigh.[5] The method of assessing fatigue level of any one of [1] to [3],wherein the method comprises assessing the subject to be in a state ofchronic fatigue caused by a disease when the amount of human herpesvirusis high.[6] The method of assessing fatigue level of any one of [1] to [5],wherein the fatigue level is the level of medium- or long-term fatiguecaused by everyday life or fatigue caused by a disease, and mentalfatigue and/or complex fatigue.[7] The method of assessing fatigue level of any one of [1] to [6],wherein the body fluid is at least one fluid selected from blood,saliva, cerebrospinal fluid, and urine.[8] The method of assessing fatigue level of any one of [1] to [7],wherein the herpesvirus is at least one virus selected from humanherpesvirus 6, human herpesvirus 7, human cytomegalovirus, andEpstein-Barr virus (EBV).[9] A kit for assessing fatigue level, wherein the kit is for carryingout the method of assessing fatigue level of any one of [1] to [8].[10] The kit of [9], comprising a means for measuring the amount of HHVin a collected body fluid, and which carries an indication that the kitcan be used to assess fatigue level of a test subject using humanherpesvirus amount in a body fluid as an indicator.[11] A method of assessing an anti-fatigue effect of a candidateanti-fatigue substance, wherein the method comprises:(i) a pre-intake HHV amount-measuring step, which comprises obtaining abody fluid from a test subject before the test subject takes thecandidate anti-fatigue substance and measuring the amount of HHV in thebody fluid;(ii) a post-intake HHV amount-measuring step, which comprises obtaininga body fluid from the test subject after the test subject takes thecandidate anti-fatigue substance and measuring the amount of HHV in thebody fluid;(iii) a change-in-virus-amount-calculating step, which comprisescalculating a change in an HHV amount in a body fluid before and afterthe intake of the candidate anti-fatigue substance, based on themeasurement result on a change in an HHV amount before and after theintake of the candidate anti-fatigue substance, which result is obtainedthrough the pre-intake HHV amount-measuring step of (i) and thepost-intake HHV amount-measuring step of (ii); and(iv) an anti-fatigue potency-measuring step, which comprises measuringan in vivo anti-fatigue potency of the candidate anti-fatigue substancebased on the change in the HHV amount in the body fluid before and afterthe intake of the candidate anti-fatigue substance, which change isobtained by the change-in-virus-amount-calculating step of (iii).[12] A method of assessing an anti-fatigue effect of a candidateanti-fatigue substance, wherein the method comprises:(i) an HHV amount-measuring step, which comprises obtaining a body fluidfrom a test subject who has taken the candidate anti-fatigue substanceand from a test subject who has not taken the candidate anti-fatiguesubstance, and measuring the HHV amount in each body fluid;(ii) a change-in-virus-amount-calculating step, which comprisescalculating a change in an HHV amount in a body fluid depending onwhether or not the candidate anti-fatigue substance has been taken,based on the measurement result on a change in an HHV amount dependingon whether or not the candidate anti-fatigue substance has been taken,which result is obtained through the process for HHV amount-measuringstep of (i), and(iii) an anti-fatigue potency-measuring step, which comprises measuringan in vivo anti-fatigue potency of the candidate anti-fatigue substancebased on the change in the amount of HHV in a body fluid depending onwhether or not the candidate substance for an anti-fatigue substance hasbeen taken, which result is obtained through thechange-in-virus-amount-calculating step of (ii).[13] The method of assessing an anti-fatigue effect of [11] or [12],wherein the method comprises assessing the anti-fatigue effect of thecandidate anti-fatigue substance using the method of assessing fatiguelevel of any one of [1] to [8] or by using the kit for assessing fatiguelevel of any one of [9] to [10].[14] The method of assessing an anti-fatigue effect of a candidateanti-fatigue substance of any one of [11] to [13], wherein the method isa method of screening for an anti-fatigue substance.

EFFECTS OF THE INVENTION

The present invention's methods of assessing fatigue level, kits forassessing fatigue level, methods of application thereof, and methods ofassessing anti-fatigue potency of anti-fatigue substances produce theeffect of enabling the quantitative assessment of fatigue level of testsubjects by using body fluid obtained from the subjects. Furthermore,the methods and kits of the present invention are not just simple andeasy, but they also do not restrain the subjects for a long period, andtherefore do not cause pain or hassle to the test subjects. The methodsand such are also simple and easy for a person implementing them and areeffective in that they are very easy to handle for both the testsubjects and implementers. Therefore, they are very useful techniquesapplicable to methods of screening for anti-fatigue substances and an invivo assessment of food products and such advertising anti-fatiguepowers.

The kits for assessing fatigue level of the present invention enable theassessment of the effect and efficacy of medicines and food productshaving an effect of preventing or relieving fatigue, for example, byobtaining a body fluid from test subjects to determine and calculate theamount of HHV in the body fluid. In other words, an effect and efficacyof medicines or food products having a fatigue-preventing orfatigue-relieving effect within a living body can be simply, easily, andquantitatively determined.

The present invention's methods enable the simple, easy, reliable, andeven quantitative measurement as to what extent an anti-fatiguesubstance is effective in improving human fatigue symptoms, that is, theanti-fatigue potency of the anti-fatigue substance.

The present invention provides methods and kits for simply, easily andquantitatively measuring and assessing the degree of fatigue in everydaylife, as well as methods of application thereof. Thus, according to thepresent invention, one can objectively know the fatigue degree ineveryday life and can avoid the occurrence of various diseases caused byunconsciously accumulated fatigue. Furthermore, the incidence of deathfrom overwork, which is caused by continuing the overwork unconscious ofthe fatigue, can be decreased.

Furthermore, the present invention can provide consumers and societywith information on the extent of the in vivo anti-fatigue potency ofmedicines and food products supplied abundantly in the market, whichadvertise fatigue-relieving effects, nutritional fortification, andnutritional support. Such information can be used as a guide forconsumers to select anti-fatigue foods and medicines effective inpreventing overfatigue, or those effective as nourishing tonics. Inthese points, the present invention is very useful and has a strongsocial impact.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schedule of the test for measuring the amount of HHVexpression in Example 1.

FIG. 2 depicts the measurement results of HHV expression levels inExample 1, which are displayed as a graph showing the changes in thetest subjects' HHV-6 expression rate (proportion (%) of the testsubjects expressing HHV-6 DNA among total test subjects). Each of theexpression rates on the first test day (on exertion) and on the secondtest day (immediately after holiday) is shown.

FIG. 3 depicts the results of measurement of the amounts and frequencyof HHV DNA expression in Example 2, which are displayed as a graphshowing the changes in the amount and frequencies of HHV-7 DNAexpression in CFS patients and healthy individuals. The vertical axisshows the expressed amount of HHV-7 DNA, and each column showsindividual test subjects for whom positive expression was observed.

BEST MODE FOR CARRYING OUT THE INVENTION

Herein below, the present invention's methods and kits for assessingfatigue level and the methods of application thereof will be explained;however, the present invention is not limited thereto.

“Fatigue” in the present application refers to a tentative phenomenonobserved when physical or mental burden is continuously given, and inwhich phenomenon, physical and/or mental functions and physical strengthare qualitatively or quantitatively deteriorated. “Fatigue” isclassified into physiological fatigue and pathological fatigue.“Physiological fatigue” in the present application means a tentativephenomenon observed when a healthy person is given continuous physicalor mental burden, and in which phenomenon, physical and mental workcapability is qualitatively or quantitatively deteriorated.“Pathological fatigue” means fatigue accompanying diseases such aschronic fatigue syndrome, mental disorders, heart disease, hepatitis,anemia, various infectious diseases, and malignant tumors.

“Physiological fatigue” is further classified into “acute fatigue” and“chronic fatigue”. “Acute fatigue” in the present application meanstransient fatigue recoverable after a proper rest. “Chronic fatigue”means fatigue lasting for a long period of time due to the accumulationof fatigue as a result of incomplete day-to-day recovery. Medium- orlong-term fatigue according to the present application means acutefatigue preceding the above-mentioned chronic fatigue.

“Acute fatigue” and “chronic fatigue” are each classified into “mentalfatigue” and “physical fatigue”. “Mental fatigue” in the presentapplication means not only fatigue caused by complicated calculationsand/or memorization or by mental activities like thinking; also impliedis fatigue including eyestrain and/or mental stress caused whenexcessive emotional activity or willpower, including patience and/orstrain or frustrations arising from working under time constraints, arerequired. “Physical fatigue” in the present application means fatiguecaused by carrying out physical work or fatigue due to effects ofelectromagnetic waves. Furthermore, “mental fatigue load” according tothe present application means giving the above-mentioned mental fatigue.

“Prolonged (persistent) fatigue” in the present application means astate in which fatigue caused by some trigger continues for a longperiod of time without recovery.

“Complex fatigue” in the present application means fatigue comprising acombination of the above-mentioned various kinds of fatigue.

“Fatigue level (degree)” in the present application refers to thelevel/degree of a state of deterioration of physical and/or mentalfunction and physical strength. This state is caused by theabove-mentioned various “fatigues” and accompanies a unique morbid anduncomfortable feeling and a desire for resting caused by excessivephysical or mental activity, or a disease. Here, “a state ofdeterioration of physical or mental function” means qualitative orquantitative deterioration in the capability of performing physical andmental work.

A “state of overfatigue (excess fatigue)” is a condition that leads topathological fatigue and where biological rhythms collapse andlife-maintaining functions break down as a result of continuedphysiological fatigue and chronic fatigue as mentioned above.

Though the present invention targets all of the above-mentioned various“fatigues”, physiological fatigue, especially acute, medium- orlong-term fatigue is preferred. Furthermore, in the present invention,fatigue caused by a mental fatigue load is preferred among medium- orlong-term fatigues. Additionally, prolonged (persistent) fatigue andfatigue (morbid fatigue) accompanying diseases such as chronic fatiguesyndrome (CFS), cancers, heart diseases, and lifestyle-related diseasesare preferred as targets of the present invention.

Most appropriate virus conditions for carrying out the present inventioninclude being a virus that has latently infected most people allowingmany people be test targets, and being able to easily measure thereactivated virus.

Viruses fulfilling the above conditions include human herpesvirus (HHV).As mentioned above, three subfamilies exist in HHV; for instance, humanherpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7), and humancytomegalovirus (also known as human herpesvirus 5) belong to βsubfamily, and Epstein-Barr virus (EBV) belongs to γ subfamily. Amongadult Japanese, 90% or more people have already been infected with theabove viruses, and carry the viruses in a latent state in their bodies.Especially, HHV-6 and HHV-7 infect all people at the age of two or moreand five or more, respectively; therefore all adults are considered tohave latently-infected viruses in their bodies. Thus, HHV-6, HHV-7,cytomegalovirus, and Epstein-Barr virus (EBV) are considered to besuitable for the present invention.

Because the virus DNA is known to be released into saliva when theseviruses are reactivated, virus reactivation can be simply and easilydetermined by measuring virus DNA in saliva.

Herein below, an outline of the present invention will be brieflyexplained. Many parts of the outline of the methods described here arecommon to the later-described kits and methods of application.

(1) Methods for Assessing Fatigue Level:

The present inventors discovered that human fatigue level can be simply,easily, and quantitatively measured by sampling a body fluid of testsubjects to measure the amount of HHV in the body fluid. This methoddoes not require a large apparatus and needs only a short time forobtaining a body fluid; therefore the method imposes less timeconstraints on the test subjects and is very simple and easy for personsimplementing the method.

Although any virus belonging to human herpesvirus (HHV) family is usableas a virus targeted in the present invention, HHV-6, HHV-7,cytomegalovirus, and Epstein-Barr virus (EBV) are preferable.Furthermore, HHV-6 and HHV-7 are more preferable.

A body fluid targeted in the present invention may be at least one ormore kinds of body fluids selected from blood, saliva, cerebrospinalfluid, and urine, but saliva is preferable.

Methods for collecting saliva may be at least one or more kinds selectedfrom swabbing viscous fluid of the pharynx with a cotton bud, directlyspitting out saliva into a test tube, and using a saliva-sampling tool,for example a Salivette and such. A method using a Salivette ispreferable.

Treatment of the oral cavity before saliva sampling includes, forexample, resting quietly without taking food for a long period of time,and rinsing out the oral cavity with water just before saliva sampling.Rinsing out the mouth with water just before saliva sampling ispreferable.

Conventionally known methods can be used as a method for measuring theamount of HHV in a body fluid, and those skilled in the art canappropriately choose specific techniques and conditions. Methods formeasuring the amount of a virus include those measuring the amount ofviral nucleic acids and those measuring the amount of viral proteins.

As methods for measuring the amount of viral nucleic acids, included area method that purifies DNA from saliva and measures the amount of virusDNA by a PCR method with PCR primers specific for each human herpesvirus(reference: Journal of Medical Virology, 1990, 32:139-142 “Detection ofhuman herpesvirus 6 DNA in throat swabs by polymerase chain reaction”),a method for measuring by double-nested PCR (reference: Journal ofInfectious Diseases, 1993, 167:1197-1200 “Association of HHV-6 infectionof the central nervous system with recurrence of febrile convulsions”),and such. In the present invention, a double-nested PCR method or areal-time PCR method are preferable.

Methods for measuring the amount of viral protein include, for example,an immunoassay method using an antibody against a viral protein. Thesandwich ELISA method is one representative example of an immunoassay(reference: J. Clin. Microbiol. 1983 May; 17 (5):942-4. “Typing ofherpes simplex virus isolates by enzyme-linked immunosorbent assay:comparison between indirect and double-antibody sandwich techniques.”Gerna G, Battaglia M, Revello M G, Gerna M T.)

In the present invention's methods for measuring fatigue level, it ispreferable to assess a fatigue level of a subject to be high when theabove-mentioned HHV amount in a body fluid is high. This is because thatthe amount of HHV expression in a body fluid of test subjects increasesas the subject's fatigue degree rises, as shown in the Examplesdescribed later.

Furthermore, it is obvious for those skilled in the art that a part orall of the methods for assessing fatigue level according to the presentinvention can be practiced by using a conventionally known calculatingapparatus (a data-processing apparatus) such as a computer. For example,the methods for assessing fatigue degree according to the presentinvention can be said to comprise a measurement process in which theamount of HHV in a body fluid is measured and an assessment process inwhich fatigue level of a subject is assessed based on the measurementresults of HHV amount in the body fluid. Among these processes, acalculating apparatus is especially utilizable in the assessmentprocess.

(2) Kits for Assessing Fatigue Level:

Next, the present invention's kits for assessing fatigue level will beexplained. The kits for assessing fatigue level according to the presentinvention are for assessing fatigue level in humans. In other words, anykits for carrying out the present invention's methods for assessingfatigue level as explained in the above section (1) are included. Inmore detail, the kits can be those comprising a means for measuring theamount of HHV in a body fluid of test subjects. As a means for measuringthe amount of HHV in a body fluid according to the present invention,any means necessary to practice conventionally known measurement methodscan be used. Specifically, a means necessary to practice conventionallyknown measurement methods includes, for example, reagents, apparatuses,equipments, catalysts, and such that are necessary to carry out themethods for measuring the amount of HHV in a body fluid as explained inthe above section (1). The kits for assessing fatigue degree accordingto the present invention may comprise a means for obtaining body fluidof test subjects. Labels on the packaging material of the kits ordocuments attached to them may indicate that the kits can be used toassess fatigue level of test subjects by using human herpesvirus (HHV)amount in a body fluid as an indicator.

Furthermore, the kits for assessing the degree of fatigue according tothe present invention may be kits comprising a conventionally knowncalculating apparatus such as a computer.

(3) Applications of the Methods and Kits for Assessing Fatigue:

As mentioned above, the methods and kits for assessing fatigue levelaccording to the present invention enable the quantitative measurementand assessment of the anti-fatigue potency of an anti-fatigue substancein the body of a test subject by measuring the amount of HHV in a bodyfluid of the test subject before and after the test subject takes theanti-fatigue substance. Furthermore, such methods and kits are bothsimple and easy, and also do not require a large apparatus or long timeconstraints; therefore they are advantageous in being easy in handlingfor both test subjects and operators.

Therefore, the present invention also includes methods for measuring theanti-fatigue potency of an anti-fatigue substance by using either themethods or kits for assessing fatigue level according to the presentinvention. Such methods for measuring the anti-fatigue potency of ananti-fatigue substance can be said to comprise, for example, thefollowing processes:

(i) a pre-intake HHV amount-measuring step, which comprises obtaining abody fluid from a test subject before the test subject takes thecandidate anti-fatigue substance and measuring the amount of HHV in thebody fluid;(ii) a post-intake HHV amount-measuring step, which comprises obtaininga body fluid from the test subject after the test subject takes thecandidate anti-fatigue substance and measuring the amount of HHV in thebody fluid;(iii) a change-in-virus-amount-calculating step, which comprisescalculating a change in an HHV amount in a body fluid before and afterthe intake of the candidate anti-fatigue substance, based on themeasurement result on a change in an HHV amount before and after theintake of the candidate anti-fatigue substance, which result is obtainedthrough the pre-intake HHV amount-measuring step of (i) and thepost-intake HHV amount-measuring step of (ii); and(iv) an anti-fatigue potency-measuring step, which comprises measuringan in vivo anti-fatigue potency of the candidate anti-fatigue substancebased on the change in the HHV amount in the body fluid before and afterthe intake of the candidate anti-fatigue substance, which change isobtained by the change-in-virus-amount-calculating step of (iii).

Furthermore, the present invention's methods for measuring theanti-fatigue potency of a candidate substance for an anti-fatiguesubstance (hereafter “candidate anti-fatigue substance”) can be carriedout as methods in which the above-mentioned methods for measuringanti-fatigue potency is applied to test subjects administered with thecandidate substance for an anti-fatigue substance (administered group),and to test subjects not administered with the substance(non-administered group).

One of ordinary skill in the art can appropriately choose the method ofintake by which test subjects take the candidate anti-fatigue substance.“Intaking” in the present application means not only the test subject'singestion of the substance as a food and drink, but also the oral orparenteral administration of the substance.

Being “anti-fatigue” according to the present application means havingan effect of relieving, suppressing, or preventing fatigue.“Anti-fatigue substances” include substances having the effect torelieve, suppress, or prevent fatigue, and substances supplementingbiological functions. “Substances supplementing biological functions”mean substances that adjust the balance in the body and comprisesubstances or such that have a function of enhancing an immune function.

The present invention's methods and kits for assessing fatigue level areapplicable, for example, to methods of screening for an anti-fatiguesubstance. In other words, the methods of screening for an anti-fatiguesubstance according to the present invention can be any method as longas it utilizes either an above-mentioned method for assessinganti-fatigue level or a kit for assessing anti-fatigue level to screenfor an anti-fatigue substance, and the specific methods and conditionsare not restricted.

The above-mentioned screening methods enable simple, easy, and objectiveselection of food products that actually show a good anti-fatigueactivity in vivo, for example, by orally administering to test subjectsa food product that is likely to be applicable as an anti-fatigue food.Therefore, an anti-fatigue substance or anti-fatigue food selected bythe above-mentioned screening methods has a proved in vivo effect andwill be able to obtain a high appraisal in the market.

Novel anti-fatigue substances according to the present invention can beany substance obtained by the above screening methods, and anti-fatiguesubstances obtained by the above screening methods are also included inthe present invention.

As fatigue turned into a social problem, the types and amounts ofanti-fatigue substances and anti-fatigue food products advertisinganti-fatigue functions have increased, strongly requiring thedevelopment of a method for appropriately assessing their anti-fatiguepotency. These needs can be addressed by utilizing the presentinvention's methods and kits for assessing fatigue level and theirmethods of application.

Herein below, modes for carrying out the present invention will beexplained in more detail by using examples based on the attacheddrawings. Needless to say, the present invention is not restricted tothe following examples, and details can obviously include variations.Furthermore, the present invention is not restricted to theabove-mentioned embodiments. Various modifications are possible, andembodiments obtainable by appropriately combining each disclosedtechnical means are also comprised within the technical scope of thepresent invention.

The present invention is a product of the research project“Molecular/neural mechanisms of fatigue and fatigue sensation and thedevelopment of the way to overcome fatigue”, in “Studies correspondingto needs of people” funded through the Special Coordination Funds forPromoting Science and Technology of the Ministry of Education, Culture,Sports, Science and Technology (MEXT), Japan.

All prior art references cited herein are incorporated by reference.

Example 1

Targeting healthy individuals, the expression of HHV-6 DNA in saliva oftest subjects during work and before and after rest was measured. Thenumber of target test subjects was 20 (eleven males and nine females),and the average age was 34.45±8.92. The present Example was conductedusing samples obtained from the test subjects participating in thepresent study, after fulfilling informed consent conditions.

(1) Measurement of Fatigue Level (1-1) Test Schedule

Saliva sampling from test subjects was carried out according to the testschedule shown in FIG. 1. During the resting period, the test subjectsdid not do any work, and avoided excessive exercise.

(1-2) Method of Saliva Sampling

Test subjects were fasted from nine in the evening of the day prior tothe test day. The test subjects rinsed the oral cavity twice withdistilled water just before saliva sampling, which was done by holdinginner cotton of a Salivette (SARSTEDT AG & Co.) in the oral cavity fortwo minutes.

(1-3) Method for Sample Preservation

Sampled saliva was preserved by freezing (−80° C.) on the same day.

(1-4) Purification of DNA

Purification of DNA from the collected saliva samples was carried out inthe presence of carrier RNA using a commercially available kit for DNApurification (QIAamp MinElute Virus Spin Kit, QIAGEN).

(1-5) Measurement of the Amount of Viral DNA of HHV

The measurement of the amount of herpesvirus DNA was carried out byusing a double-nested PCR method with PCR primers specific for each ofthe four species HHV-6, HHV-7, human cytomegalovirus, and Epstein-Barrvirus (EBV). Among HHV-6-specific PCR primer sequences, the sequences ofthe first PCR primer are shown in SEQ ID NOs: 1 and 2, and the sequencesof the second PCR primer are shown in SEQ ID NOs: 3 and 4. AmongHHV-7-specific PCR primer sequences, the sequences of the first PCRprimer are shown in SEQ ID NOs: 5 and 6, and the sequences of the secondPCR primer are shown in SEQ ID NOs: 7 and 8. Among humancytomegalovirus-specific PCR primer sequences, the sequences of thefirst PCR primer are shown in SEQ ID NOs: 9 and 10, and the sequences ofthe second PCR primers are shown in SEQ ID NOs: 11 and 12. AmongEpstein-Barr virus (EBV)-specific PCR primer sequences, the sequences ofthe first and the second PCR primers are shown in SEQ ID NOs: 13 and 14.The same primer sequences were used in the first and the second PCR forEpstein-Barr virus (EBV).

A reference for the measurement of the amount of HHV-6 DNA:Journal of Virol. 2002, 76(8): 4145-4151, K. Kondo et al.“Identification of human herpesvirus 6 latency-associated transcripts.”A reference for the measurement of the amount of human cytomegalovirusDNA:Proc. Natl. Acad. Sci. USA 1996, 93: 11137-11142, K. Kondo et al. “Humancytomegalovirus latent gene expression in granulocyte-macrophageprogenitors in culture and in healthy seropositive individuals.”

(2) Results

Among the above four species of viruses, HHV-6 and HHV-7 were found tobe frequently detected in saliva of healthy adults. On the other hand,the detection frequency for human cytomegalovirus and Epstein-Barr viruswas lower than that for HHV-6 and HHV-7.

The measurement results on the amount of HHV-6 DNA are shown in FIG. 2.For one measurement, 20 μl saliva was used. HHV-6 DNA was detected in88% of the test subjects engaged in moderately excessive work due toprolonged working time and such (the first test day). In contrast, HHV-6DNA was detected in 23.8% of the test subjects immediately after aholiday (the second test day). These results show that HHV-6 issignificantly reactivated on exertion. These results led to thediscovery that HHV-6 DNA expressed in saliva through the reactivation ofHHV-6 is a fatigue biomarker (a biological index factor) that variesaccording to fatigue. Accordingly, an objective method for assessingfatigue degree by detecting HHV-6 DNA released into saliva as a resultof the reactivation of HHV-6 was developed, enabling a simple and easyassessment of fatigue degree.

Example 2 (3) The amount of HHV-7 expression in chronic fatigue syndrome(CFS) patients (3-1) Testing Methods

Twenty-four chronic fatigue syndrome (CFS) patients and the twentyhealthy individuals of Example 1 were targeted. As the patients, CFSpatients attending the CFS outpatient unit of Osaka University Hospitalwho fulfilled the diagnostic criteria of the Ministry of Health, Labourand Welfare were targeted. The present Example was conducted usingsamples obtained from the test subjects participating in the presentstudy, after fulfilling informed consent conditions.

(3-2) Method of Saliva Sampling

Subjects rinsed the oral cavity twice with distilled water just beforesaliva sampling, which was done by holding inner cotton of a Salivette(SARSTEDT AG & Co.) in the mouth cavity for two minutes.

(3-3) Method of Detection

The detection and measurement of HHV were done in the same manner as initems (1-3) and (1-5) of Example 1.

(4) Results

Among the above-mentioned four species of viruses, HHV-7 was especiallyfound to be frequently and abundantly detected in saliva of patients. Onthe other hand, the detection frequency of human cytomegalovirus, HHV-6,and Epstein-Barr virus was lower compared to that of HHV-7.

The measurement results of HHV-7 are shown in FIG. 3. The amount ofHHV-7 DNA was semi-quantitatively measured by conducting a double-nestedPCR method after serially diluting 20 μl of saliva. HHV-7 DNA wasdetected in 92% of CFS patients among the test subjects. In contrast, inhealthy individuals, HHV-7 DNA was detected in 50% of test subjectsduring work and in only 30% of test subjects at rest. The amount ofHHV-7 DNA in half of the CFS patients reached an amount 10 to 100 foldlarger than the average amount detected in healthy persons. Theseresults show that HHV-7 is significantly reactivated in the chronicfatigue state that accompanies disease. These results led to thediscovery that HHV-7 DNA expressed in saliva due to reactivation ofHHV-7 is a fatigue biomarker (a biological index) that varies accordingto chronic fatigue caused by diseases or such. Accordingly, an objectivemethod for assessing the degree of fatigue by detecting HHV-7 DNAreleased into saliva due to reactivation of HHV-7 was developed,enabling simple and easy assessment of decline in physical strengthcaused by chronic fatigue.

INDUSTRIAL APPLICABILITY

The methods for assessing fatigue level according to the presentinvention can be used to elucidate mechanisms of stress and/or fatigue.The methods can also be used to develop stress-relieving methods, toassess the extent of fatigue, and to assess the decline in physicalstrength that accompanies a disease. By applying the present invention,it is possible to quantify (assess) the effect of health foods, foodsfor specified health uses, nutrition-supplement drinks, and substancessupplementing biological functions in the market that advertiseanti-fatigue activity. Accordingly, the present invention is applicableto a broad range of fields such as medical services, drug manufacturing,health food industry, health equipment industry, and such.

1-14. (canceled)
 15. A method of assessing if a test subject is in astate of physiological fatigue comprising: (a) preparing a saliva samplefrom a test subject; (b) measuring the amount of human herpesvirus 6 or7 viruses in the saliva sample by measuring the amount of humanherpesvirus 6 or 7 protein present in the sample; and (c) determining ifthe amount of either human herpesvirus 6 or 7 in the saliva sample ishigher than the average amount detected in healthy persons whosephysical and mental work capability is not deteriorated, wherein ahigher than average amount of either human herpesvirus 6 or 7 in salivasignifies that the test subject is experiencing a reactivated humanherpesvirus 6 or 7 viral infection and wherein the higher than averageamount of either human herpesvirus 6 or 7 in saliva is a positiveindication of physiological fatigue.
 16. The method of claim 15, whereinthe physiological fatigue is due to an accumulation of physiologicalfatigue arising from everyday life.
 17. The method of claim 15, whereinthe amount of human herpesvirus 6 or 7 protein present in the sample ismeasured by an immunoassay method.
 18. A method of assessing if a testsubject is in a state of physiological fatigue comprising: (a) preparingsaliva samples from healthy adults immediately after resting period; (b)preparing a saliva sample from the test subject at an arbitrary timepoint; (c) measuring the amount of human herpesvirus 6 or 7 in thesamples obtained in (a) and (b) by measuring the amount of humanherpesvirus 6 or 7 protein present in the samples; and (d) assessingthat the test subject is in a state of physiological fatigue when theamount of human herpesvirus 6 or 7 in the sample obtained in (b) isgreater than the average amount of human herpesvirus 6 or 7 in thesample obtained in (a).
 19. The method of claim 18, wherein thephysiological fatigue is due to an accumulation of physiological fatiguearising from everyday life.
 20. The method of claim 18, wherein theamount of human herpesvirus 6 or 7 protein present in the sample ismeasured by an immunoassay method.
 21. A method of assessing thephysiological fatigue level of a test subject comprising: (a) preparingsaliva samples from a test subject immediately after resting period; (b)preparing a saliva sample from the test subject of (a) at an arbitrarytime point; (c) measuring the amount of human herpesvirus 6 or 7 in thesamples obtained in (a) and (b) by measuring the amount of humanherpesvirus 6 or 7 protein present in the samples; and (d) assessingthat the physiological fatigue level of the test subject is high whenthe amount of human herpesvirus 6 or 7 in the sample obtained in (b) isgreater than the average amount of human herpesvirus 6 or 7 in thesample obtained in (a).
 22. The method of claim 21, wherein thephysiological fatigue is due to an accumulation of physiological fatiguearising from everyday life.
 23. The method of claim 21, wherein theamount of human herpesvirus 6 or 7 protein present in the sample ismeasured by an immunoassay method.
 24. A method of assessing theincrease or decrease in the physiological fatigue level of a testsubject comprising: (a) preparing a base line saliva sample from a testsubject; (b) preparing a saliva sample from a test subject of (a) at atime point different from when the base line sample of (a) is obtained;(c) measuring the amount of human herpesvirus 6 or 7 in the samplesobtained in (a) and (b) by measuring the amount of human herpesvirus 6or 7 protein present in the samples; and (d) assessing that thephysiological fatigue level of the test subject at the time point,wherein if the amount of human herpesvirus 6 or 7 in the sample obtainedin (b) is significantly greater than the amount of human herpesvirus 6or 7 in the sample obtained in (a), then the physiological fatigue levelis increased and wherein if the amount of human herpesvirus 6 or 7 inthe sample obtained in (b) is significantly less than the amount ofhuman herpesvirus 6 or 7 in the sample obtained in (a), then thephysiological fatigue level is decreased.
 25. The method of claim 24,wherein the physiological fatigue is due to an accumulation ofphysiological fatigue arising from everyday life.
 26. The method ofclaim 24, wherein the amount of human herpesvirus 6 or 7 protein presentin the sample is measured by an immunoassay method.
 27. A method ofassessing the physiological fatigue level of a test subject comprising:(a) preparing a saliva sample from a healthy adult immediately afterresting period; (b) preparing a saliva sample from the test subject atan arbitrary time point; (c) measuring the amount of human herpesvirus 6or 7 in the samples obtained in (a) and (b) by measuring the amount ofhuman herpesvirus 6 or 7 protein present in the samples; and (d)assessing that the physiological fatigue level of the test subject ishigher than the physiological fatigue level of the healthy adultimmediately after resting period when the amount of human herpesvirus 6or 7 in the sample obtained in (b) is significantly greater than theamount of human herpesvirus 6 or 7 in the sample obtained in (a). 28.The method of claim 27, wherein the physiological fatigue is due to anaccumulation of physiological fatigue arising from everyday life. 29.The method of claim 27, wherein the amount of human herpesvirus 6 or 7protein present in the sample is measured by an immunoassay method. 30.A method of assessing the physiological fatigue level of a test subjectcomprising: (a) preparing a saliva sample from a test subjectimmediately after resting period; (b) preparing a saliva sample from thetest subject at an arbitrary time point; (c) measuring the amount ofhuman herpesvirus 6 or 7 in the samples obtained in (a) and (b) bymeasuring the amount of human herpesvirus 6 or 7 protein present in thesamples; and (d) assessing that the physiological fatigue level of thetest subject at the time point when the sample obtained in (b) at anarbitrary time point is higher than the physiological fatigue level ofthe test subject immediately after resting period when the amount ofhuman herpesvirus 6 or 7 in the sample obtained in (b) is significantlygreater than the amount of human herpesvirus 6 or 7 in the sampleobtained in (a).
 31. The method of claim 30, wherein the physiologicalfatigue is due to an accumulation of physiological fatigue arising fromeveryday life.
 32. The method of claim 30, wherein the amount of humanherpesvirus 6 or 7 protein present in the sample is measured by animmunoassay method.